Understanding hypermucoviscosity in Klebsiella

Abstract Klebsiella pneumoniae (Kpn) is a leading cause of Gram-negative nosocomial infections and is associated with a high mortality rate. Antibiotic resistance is a growing issue among the Enterobacteriaceae and of the Enterobacteriaceae Kpn is the most prevalent extended spectrum β-lactamase and carbapenem resistant Enterobacteriaceae isolate. The increasing prevalence of antibiotic-resistant Kpn only serves to compound its clinical importance and to complicate treatment options. Capsule has been established as a key virulence factor and is this species best studied virulence factor. Kpn strains are broadly classified as hypervirulent (hv) or classical, with hv strains typically causing community acquired liver abscess and invasive infections. Classical strains are more typically associated with nosocomial infections. The hv strains have a hypermucoviscous (HMV) phenotype thought to be due to over-production of capsule and have acquired rmpA that contributes to increased capsule (cps) gene expression. Most classical strains, including recent clinical isolates associated with carbapenem resistance (ST258) are not HMV, do not have the rmpA gene and are not virulent in mouse models of infection. Recent reports of these multidrug resistant strains acquiring the HMV phenotype is of significant concern and amplifies the need to understand what HMV is, how it is produced and how it contributes to virulence. Despite the apparent association between HMV and over-production of capsule, what causes the HMV phenotype and its relationship to capsule production is not known. Recent work from our lab using the HMV strain KPPR1S has shown that deletion of rmpA causes decreases in expression from cps promoters, and reduction (but not complete loss) in both capsule production and HMV. In addition, we found that rmpA is the first gene of an operon (rmpADC) and that RmpA positively regulates expression of the operon. Results from analysis of a complete deletion of the operon with the individual genes indicates that from this locus, RmpA primarily is required for expression of the operon, that only RmpC is necessary for expression of cps genes, and that only RmpD is necessary for HMV. Consistent with this, a mutant lacking only rmpD retains WT levels of cps gene expression, capsule production and is HMV negative. The rmpD mutant is the first mutant identified that separates HMV from the over- production of capsule, and the phenotype of the strain only expressing rmpC indicates that over- production of capsule alone is not sufficient for HMV. Due to the importance of HMV to the virulence of hv Kpn strains, the potential for classical strains to acquire HMV, the incomplete picture of what constitutes HMV and what is required to produce HMV, we propose to a) gain a better understanding of RmpD, (b) characterize the interacting partners of RmpD and how they affect HMV, and determine the composition of the HMV exopolysaccharide, and (c) examine how HMV contributes to virulence.